DNA Fingerprinting and Japanese Knotweed
DNA FINGERPRINTING
&
JAPANESE
KNOTWEED
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DNA Fingerprinting and Japanese Knotweed
DNA FINGERPRINTING AND JAPANESE KNOTWEED
DNA,
or deoxyribonucleic acid, is found in all living
organisms. DNA is a long chain of nucleotides, the order of
which differs from organism to organism.
In complex
organisms such as humans and other mammals, each
individual (except for identical twins) has unique DNA.
Differences in DNA make one individual different from the
next – for example, one person might have DNA containing
genes for blue eyes, while another has DNA containing genes
for brown eyes.
DNA fingerprinting is a scientific technique that can provide us
with information about an organism’s DNA. In DNA fingerprinting, DNA is firstly
cut into smaller pieces by enzymes called restriction endonucleases, which
recognise specific sequences of bases within the DNA molecule. As DNA from
each organism is different, these restriction endonucleases will cut the DNA from
each individual at different places and produce fragments of different lengths.
Gel electrophoresis is then used to separate the DNA fragments. To do this, the
pieces of DNA are placed in a gel, and an electric current is applied to the gel.
The electric current makes the DNA fragments move through the gel, with the
negatively charged DNA moving towards the positive electrode.
Smaller
fragments move more easily through the gel and so travel faster than larger ones.
The DNA fragments create many different bands on the gel and form a banding
pattern representative of an individual. The banding patterns from different DNA
samples can then be compared to see if the DNA came from the same or related
individuals. For more information on DNA fingerprinting and its applications in a
forensics context, go to:
www.protist.biology.washington.edu/fingerprint/dnaintro.html
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DNA Fingerprinting and Japanese Knotweed
You might have heard of the use of DNA fingerprinting to identify criminals, test
for paternity and diagnose genetic diseases.
But DNA fingerprinting can also be
an invaluable tool to scientists who study plants and animals, and conservationists
trying to save endangered plants and animals. DNA fingerprinting can be used to
explore genetic diversity, determine new species, and understand movement of
organisms within their environment, to name just a few uses. Today you will
learn how to use DNA fingerprinting to better understand the natural world.
The family history of Japanese Knotweed
Japanese Knotweed (Fallopia japonica) is a plant native to Japan, Taiwan and
Northern China.
It was introduced into the UK in the 1850s for use as an
ornamental garden plant.
However it quickly escaped from cultivation and
rapidly started to colonise areas such as canal sides, road verges, cemeteries,
streams and river banks. Its growth in urban areas has caused significant damage
as its shoots are able to push through asphalt and damage pavements and car
parks. Also the height to which Japanese Knotweed can grow (3 metres) can
cause visibility problems on roadsides. Once this plant has established itself it is
extremely difficult to eradicate and hundreds of thousands of pounds are spent
each year on the control of this weed. In 1981 it became a criminal offence to
knowingly plant Japanese Knotweed in the wild.
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DNA Fingerprinting and Japanese Knotweed
So how does this plant manage to propagate itself so efficiently? In Britain,
Japanese Knotweed plants are all female plants (they have both male and female
sex organs but the male parts are sterile, in
Japanese Knotweed
effect making them females).
Therefore
Japanese Knotweed cannot reproduce by
standard sexual reproduction and, in Britain,
seems
incapable
of
producing
seed.
However, Japanese Knotweed has a vigorous
Photo courtesy of Michelle Hollingsworth
and fast growing root system and is capable
of regenerating a whole plant from a tiny fragment of root. It is this capability
that is thought to explain how this plant has become such a problematic invasive
weed. As sexual reproduction is one means by which genetic variation can be
maintained, the lack of sexual reproduction in this plant must impact the genetic
diversity of Japanese Knotweed in this country.
Dr M Hollingsworth from the Royal Botanic Garden in
Edinburgh has been researching the genetic diversity
in Japanese Knotweed.
Japanese Knotweed growing
through a pavement.
She has been using DNA
profiling technologies to investigate whether the
Japanese Knotweed in Britain is multiclonal or
monoclonal. If it is multiclonal, that would mean that
there are many different populations of Japanese
Knotweed in Britain.
plants
would
populations,
be
Within each population the
genetically
however,
there
similar.
would
be
Between
genetic
differences. If it is monoclonal then all the Japanese
Photo
courtesy
of Environment and
© Find
Out
Heritage Service, Cornwall.
Knotweed in Britain is genetically identical i.e. the plants are all clones of one
original plant! For more information about this research download the Leicester
University Bulletin, May 1999 (www.le.ac.uk/bulletin). The article is on page 10.
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DNA Fingerprinting and Japanese Knotweed
Today you will use a simplified version of DNA Fingerprinting to investigate
whether British Japanese Knotweed is multiclonal or monoclonal.
Knotweed is found in many areas of the British Isles.
Japanese
The map in picture 1
indicates where the Japanese Knotweed DNA samples you will be analysing today
were collected from. You must now examine the DNA profiles of these plants and
decide for yourself whether Japanese Knotweed is monoclonal or multiclonal.
Picture 1 – Map showing distribution of Japanese Knotweed in the British Isles and
where the DNA samples were collected from.
1
4
2
3
5
6
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DNA Fingerprinting and Japanese Knotweed
STUDENT GUIDE
Materials
Per individual or group
EcoR1/Pst1 enzyme mix (ENZ)
Pipette tips
P20 micropipette
Microtubes
Marker pen
Disposal jar
Foam microtube rack
Ice container
Loading dye (LD)
To be shared
DNA from location 1
DNA from location 2
DNA from location 3
DNA from location 4
DNA from location 5
DNA from location 6
HindIII DNA markers (M)
Water bath at 37°C
Agarose gel electrophoresis tanks
Power supply
TAE Electrophoresis buffer
Water
Safety
Electrical hazard from electrophoresis tank.
DNA Stain can mark clothes and be an irritant.
Eating and drinking are not allowed in the lab.
Methods
1.
Make sure your enzyme mix is kept on ice.
2.
You have been provided with labelled microtubes each containing 10µl DNA
from the different locations shown in picture 1. Label each tube with your
initials.
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DNA Fingerprinting and Japanese Knotweed
L1:
Location 1
L2:
Location 2
L3:
Location 3
L4:
Location 4
L5:
Location 5
L6:
Location 6
3.
Using a separate tip for each sample, pipette 10µl enzyme mix (ENZ) into
the bottom of each tube.
4.
Close the cap. Mix the enzyme and DNA by flicking the tubes gently.
5.
Incubate for 45 minutes at 37°C.
The DNA is being cut into fragments by the restriction endonucleases.
6.
Using a separate tip, add 5µl Loading Dye (LD) to each tube.
The Loading Dye is dense so it helps the DNA to sink into the wells. It also
contains a mixture of Dyes to monitor progress of the electrophoresis: a
faster moving dye which will move with DNA fragments of ~500 base pairs
and a slower moving dye which will move with DNA fragments of
approximately 5 kilo base pairs.
7.
Load 10µl of the DNA size marker (M) into the well on lane 1.
8.
Load 20µl of L1, L2, L3, L4, L5 and L6 into the wells on lanes 2-7
respectively.
9.
Close the electrophoresis tank, run at 100V for 30 minutes.
The negatively charged fragments of DNA will separate according to size.
10. Turn off the power.
11. Carefully, transfer the gel to a staining tray.
12. Cover the gel with 100x Fast BlastTM DNA stain and leave for 3 mins.
13. Pour off the stain, rinse the gel with tap water and cover with distilled
water to destain the gel, changing the water occasionally.
14. Observe the banding pattern. When bands are clearly visible drain off the
water and place the gel in a plastic bag. The gel will last for some weeks or
longer if stored in a fridge.
15. Draw the pattern of bands you see (next page).
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DNA Fingerprinting and Japanese Knotweed
RESULTS
Below, draw the pattern of bands you see on your gel.
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DNA Fingerprinting and Japanese Knotweed
Analysis Questions:
(a) From your results is Japanese Knotweed multiclonal? Explain your answer.
(b). What disadvantages are there to a plant which propagates in this way?
(c) Can you think of other uses of DNA Fingerprinting that could help scientists
research ecology or biodiversity of plants and animals?
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DNA Fingerprinting and Japanese Knotweed
TEACHER/TECHNICAL GUIDE
This scenario is designed to be used with the BIO-RAD DNA Fingerprinting Kit (Catalogue
Number 166-0007-EDU).
The instruction manual that comes with this kit contains
excellent technical and teacher materials.
We refer you to those materials for
instructions on preparing the agarose gels, enzyme mix, aliquoting of DNA samples etc.
Particular care should be taken however, to ensure that:
1) the lyophilised DNA samples and enzyme mix are thoroughly hydrated.
2) the enzymic digestion is carefully carried out, i.e. that the enzyme is well mixed
with the DNA sample and that the incubation is carried out for the full 45 minutes
at the correct temperature.
In the BIO-RAD DNA Fingerprinting scenario each DNA sample stands for a different
suspect, here (Japanese Knotweed clonality) each DNA sample stands for a different
Japanese Knotweed DNA sample collected from various regions in the British Isles. The
picture below shows the results of the DNA Fingerprinting.
In this scenario all
fingerprints are actually the same as Japanese Knotweed plants in Britain are all
genetically identical clones. Therefore only one DNA sample from the BIO-RAD kit is
actually used. Below is a table telling you which DNA sample from the BIO-RAD Kit you
could use to create this scenario.
It should be noted that the Green and Violet DNA samples (Crime Scene and Suspect 3)
are exactly the same therefore they are interchangeable. Also not all the BIO-RAD kit
DNA samples are used in this practical.
The unused DNA samples can be stored (as
directed in the instruction manual) and used at a later date.
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DNA Fingerprinting and Japanese Knotweed
Picture 1 - Result of gel electrophoresis
Table 1 - Showing DNA samples to use for each location to set up ‘Japanese Knotweed
clonality’ Scenario.
Biodiversity usage Japanese Knotweed
scenario
Location 1
Location 2
Location 3
Location 4
Location 5
Location 6
Colour Coding of BIO-RAD Usage – Location
DNA sample in Forensic scenario
on Gel
BIO-RAD kit
Yellow
Suspect 5
Lane 2
Yellow
Suspect 5
Lane 3
Yellow
Suspect 5
Lane 4
Yellow
Suspect 5
Lane 5
Yellow
Suspect 5
Lane 6
Yellow
Suspect 5
Lane 7
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DNA Fingerprinting and Japanese Knotweed
Answers to Analysis Questions
(a) From your results is Japanese Knotweed multiclonal. Explain your answer.
Answer: As all the DNA fingerprints are the same this suggests that all the Japanese
Knotweed plants in the British Isles are in fact genetically identical clones. It is not
multiclonal.
(b). What disadvantages are there to a plant which propagates in this way?
Answer: Propagating in this way means that no or little genetic variation occurs.
This means that the plant cannot evolve or respond to changes in the environment.
Other additional disadvantages for information: Any deleterious mutations which
have an effect on the growth and development of the plant cannot be removed.
Also, viruses in plants can be eliminated during a cleansing step which occurs as
part of the process of sexual reproduction (gametogenesis).
(c) Can you think of other uses of DNA Fingerprinting that could help scientists research
ecology or biodiversity of plants and animals?
Answer: Please refer to other biodiversity scenarios provided as part of this pack for
other examples. Students should be able to come up with examples of their own.
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